Composition and method for causing photodynamic damage to target cells

ABSTRACT

The present invention is directed to a composition for causing photodynamic damage to target cells comprising a photosensitiser, a photosensitiser carrier component, a component which enables target cell recognition and transport of the photosensitiser toward the interior of the target cell by specific receptor-mediated endocytosis, and a component capable of effective targeted transport of the photosensitiser within the target cells. The invention is also related to a method for causing photodynamic damage to target cells comprising the steps of: adding the composition to the cells; keeping the cells at a temperature of normal vital activity of cells with the composition for causing photodynamic damage to the target cells, said composition comprising the above-mentioned components; and exposure of the cells to light.

CROSS REFERENCES TO RELATED APPLICATIONS

This is the U.S. national phase under 35 U.S.C. §371 of InternationalApplication PCT/RU97/00018, filed Feb. 3, 1997, which claims priority toRussian Federation Application 96102402/13, filed Feb. 12, 1996.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to biotechnology, cellular and molecularbiology and, more particularly, to a method for causing photodynamicdamage to target cells which may be used in experimental medicine andpharmacology.

2. Description of Related Art

The essence of the photodynamic therapy consists in the introduction tothe organism of a photosensitiser (hereinafter referred to as “PS”)taken up practically by all cells. Due to differences inmicrocirculation, distribution, exchange intensity and so on, the PSremains a longer time in target cells, for example cancer cells, than innormal cells. If at that time, target cells and PS molecules occurringtherein are exposed to light, the latter shall cause photochemicalreactions resulting in the death of target cell.

The main side effect of the photodynamic therapy resides in an enhancedlight sensitivity of skin and eyes: patients need to be carefullyprotected against sunlight within six weeks and more. Another sideeffect consists in nausea and vomit caused in patients by theintroduction of the PS in high amounts (Photodynamic Therapy. CancerFacts, National Cancer Institute, NIH, CancerNet, 1994).

Both side effects are caused by a combination of two factors: aninsufficient PS selectivity as respects target cells, and its excessiveconcentration in the patient tissues. Improvements in the efficiency ofphotodynamic damage (hereinafter referred to as “PDD”) may be achievedby using the PS as a part of a composition.

There is a prior art composition based on microspheres was abto to enterthe cell non-specifically by phagocytosis and penetrate into lysosomes.The application of the compositions of PS with microspheres results inthe enhancement of PDD to the cell.

A drawback inherent in compositions with microspheres is that thephotodynamic action of these compositions is not specific with respectto a cell type (Bachor, R., Shea, C. R., Gillies, R., and Hasan, T.Photosensitised destruction of human bladder carcinoma cells treatedwith chlorin e₆—conjugated microspheres. Proc. Natl Acad. Sci. USA, 88,15 80-15 84, 1991).

Another prior art composition comprised chlorin e₆ with monoclonalantibodies. This prior art composition contains a component for thetarget cell identification, a PS carrier component and a PS itself. Adisadvantage with such a composition lies in its ability to act on atarget cell surface only, wherein PDD induced by the composition doesnot affect the most PDD-sensitive target cell compartments (Rakestraw,S. L., Tompkins, R. D., and Yarmush, M. L. Antibody-targeted photolysis:In vitro studies with Sn(IV) chlorin e₆ covalently bound to monoclonalantibodies using a modified dextran carrier. Proc. Natl. Acad. Sci. USA,87, 4217-4221, 1990).

This disadvantage is avoided in a composition consisting of a PS, a PScarrier component and a component for target cell recognition and PStransport toward the interior of target cells by specificreceptor-mediated endocytosis, said composition being the closest priorart composition to the present invention.

This composition is characterized by the presence of three componentsperforming the same functions: a PS, a PS carrier component and acomponent for target cell recognition and PS transport toward theinterior of the target cells by specific receptor-mediated endocytosis.

An insulin-BSA-chlorin e₆ composition, after binding to specificreceptors on the surface of target cells, has the ability ofreceptor-mediated endocytosis and, accordingly, of transport toward theinterior of cells where, after cell irradiation, the process ofgenerating active oxygen species takes place, said species being a realcytotoxic agent in inducing PDD to the cell. This composition has anadvantage over the aforementioned compositions with monoclonalantibodies or microspheres in that it is capable, through specificbinding to cell surface receptors characteristic of this cell type, ofbeing intemalised by receptor-mediated endocytosis owing to which theformation of active photooxidation products occurs inside the cells nearto damage-sensitive cell compartments. Although a number of thedrawbacks associated with known compositions are removed, thecomposition lacks any special component to direct the PS transportwithin of target cells, e.g. transport into the most sensitive cellcompartments for TDD. As a result, there is no efficient transport intothese compartments, and PDD is not as great as it might be in thepresence of said component for directing the transport inside the cells(Akhlynina, T. V., Rosenkranz, A. A., Jans, D. A., Sobolev, A. S.Insulin-mediated intracellular targeting enhances the photodynamicactivity of chlorin e₆ , Cancer Res., 55, 1014-1019, 1995).

As indicated above, side effects are caused by two reasons: aninsufficient PS selectivity as respects target cells and their excessiveconcentration in the patient tissues. Therefore, one of the directionsin the development of this subject matter resides in the enhancement tothe target cell PDD.

Enhancement of PDD to the target cell, while decreasing an acting PSconcentration, is possible by using PS derivatives having otherintracellular localisation which are most sensitive to the photodynamicaction. As alluded to above, a prior art PDD method employed PSderivatives which penetrate into the cell by non-specific intemalisationand localise in membrane components and lysosomes (Kessel, D.Determinants of photosensitisation by mono-L-aspartyl chlorin e₆ .Photochem. Photobiol., 49, 447-452, 1989). As also alluded to above, itis also possible to use PS conjugates with microspheres which enter thecell nonspecifically by phagocytosis and penetrate into lysosomes(Bachor, R., Shea, C. R., Gillies, R., and Hasan, T. Photosensitiseddestruction of human bladder carcinoma cells treated with chlorine₆—conjugated microspheres. Proc. Natl. Acad. Sci. USA, 88, 1580-1584,1991). Both known methods have the same disadvantage: PS and theirderivatives may accumulate both in target cells and normal cells, thatis to say, they are non-specific to a cell type (Aizawa, K., Okunaka,T., Kawabe, H., Yasunaka, Y., O'Hata, S., Ohtomo, N., Nishimiya, K.,Konaka, C., Kato, H., Hayata, Y., and Salto, T. Localisation ofmono-L-aspartyl chlorin e₆ (NPe₆) in mouse tissues. Photochem.Photobiol., 46, 789-793, 1987).

Improvements in the PDD specificity may be achieved by the PSconjugation with a ligand having specific receptors on the target cellsurface. For this purpose, PS compositions with monoclonal antibodies tothese cells are proposed. Such an approach enables improvements in thePDD selectivity and efficiency at the expense of a specific recognitionof target cells and binding of the compositions to the surface of thesecells.

As alluded to above, a prior art method for the target cell PDDcomprises a target cell PDD enhancement using PS—chlorin e₆ compositionswith monoclonal antibodies (Rakestraw, S. L., Tompkins, R. D., andYarmush, M. L. Antibody-targeted photolysis: In vitro studies withSn(IV) chlorin e, covalently bound to monoclonal antibodies using amodified dextran carrier. Proc. Nad. Acad. Sci. USA, 87, 4217-4221,1990): In accordance with this method, a composition comprising the PSis added to target cells, keeping the cell therewith and then exposed tolight. Due to the composition properties, the PS is subjected totargeted transport into the cells as a part of the composition, thelatter being bound to specific cell receptors. With such a method,plasma membranes are the main target for the PS. This is not optimal,however, since PDD does not extend to include much more sensitiveintracellular targets—the nucleus or lysosomes (Alper, T. CellularRadiobiology. Cambridge Univ. Press, Cambridge, 1979).

This disadvantage is avoided in the PDD method chosen by the applicantas the closest prior art method to the claimed one.

This method is characterized by the following essential features: acomposition for PDD is introduced to target cells; then keeping thecells at a temperature of normal cellular vital activity with the resultthat target cells uptake a PS as part of the composition; and the PS issubsequently photo-activated (light-irradiated). This method, however,also suffers from disadvantages consisting in that, after cells haveuptaken the PS, the latter, owing to the properties of the compositionused, follows the way predetermined by the properties of the ligand—acomponent used for the recognition and receptor-mediated endocytosis ofcells. In so doing, it will not necessarily find its way (or finds itsway to a slight extent) into other PDD-sensitive cell parts (Akhlynina,T. V., Rosenkranz, A. A., Jans, D. A., Sobolev, A. S. Insulin-mediatedintracellular targeting enhances the photodynamic activity of chlorin e₆. Cancer Res., 55, 1014-1019, 1995).

SUMMARY OF THE INVENTION

It is an object of the present invention to provide compositions andmethod for causing photodynarnic damage (PDD) to target cells to enableenhancement of target cell PDD owing to a composition capable ofeffective targeted transport of a photosensitiser (PS) within the targetcells, thereby making it possible to improve safety and economy of themethod.

This object is solved in a composition producing the target cell PDD inlow concentrations due to the fact that the composition, in addition toa PS, a PS carrier component and a component which effects target cellrecognition and receptor-mediated endocytosis, also comprises acomponent capable of effective targeted transport of the PS within thetarget cells, said composition resulting, due to the PS delivery to themost PDD-sensitive compartments, in the PDD enhancement.

BRIEF DESCRIPTION OF THE DRAWINGS

The various objects, advantages and novel features of the invention willbe more readily apparent from the following detailed description whenread in conjunction with the appended drawings, in which:

FIG. 1 shows an intracellular distribution of oxygen active speciesafter irradiation of cells which is determined by the formation of2′,7′-dichlorofluorescein. PLC/PRF/5 cells were incubated in thepresence of 100 nM (BSA)-(PI 101)-insulin-(chlorin e₆) (A) and (BSA)-(PI101T)-insulin-(chlorin e₆) (B) compositions for 18 hours at 37° C. withor without a 100-fold excess of insulin (C and D), respectively. Afterincubation, the cells were washed, incubated for 5 minutes at 37° C.with 2′,7′-dichlorofluorescein diacetate, re-washed and exposed to lightof a slide projector;

FIG. 2 shows survival rate of human hepatoma PLC/PRF/5 cells plottedagainst concentration of (P10)-insulin-(chlorin e₆) and(β-galactosidase)-insulin-(chlorin e₆) compositions and chlorin e₆ atthe irradiation dose of 12.3 kJ/m². A stands for (P10)-insulin-(chlorine₆); B stands for (β-galactosidase)-insulin-(chlorin e₆); C stands forchlorin e₆;

FIG. 3 shows survival rate of human hepatoma PLC/PRF/5 cells plottedagainst concentration of (P10)-insulin-(chlorin e₆),(β-galactosidase)-insulin-(chlorin e₆) compositions and chlorin e₆ atthe irradiation dose of 96 kJ/m². A stands for (P10)-insulin-(chlorine₆); B stands for (β-galactosidase)-insulin-(chlorin e₆); C stands forchlorin e₆;

FIG. 4 shows the comparison of localisation of oxygen active speciesafter photoactivation of compositions in human hepatoma PLC/PRF/5 cells.A stands for (P10)-insulin-(chlorin e₆; B stands for(β-galactosidase)-insulin-(chlorin e₆); C stands for(P10)-insulin-(chlorin e₆)+(human adenovirus, serotype 5, straind1-312); D stands for (β-galactosidase)-insulin-(chlorin e₆) +(humanadenovirus, serotype 5, strain d1-312). Localisation was determined bythe formation of 2′,7′-dichlorofluorescein.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, a composition for causingphotodynamic damage to target cells comprises a photosensitiser, aphotosensitiser carrier component, a component which enables target cellrecognition and transport of the photosensitiser toward the interior ofthe target cells by specific receptor-mediated endocytosis in thesecells, and a component capable of effective targeted transport of thephotosensitiser within the target cells, for example the transport intothe most PDD-sensitive compartments of these cells. Individualcomponents making up the composition may be covalently bound to eachother and/or present in a “mechanical” mixture. Said components fordirected intracellular transport represent substances which are capableof effective targeted transport of the composition or its componentsinto the nucleus. Said components having the ability of targetedtransport into the nucleus are hormones, proteins and peptides having anamino acid sequence of nuclear localisation signal (karyophilicsequence) and endosomolytic components which are capable of releasingcompositions and the PS from endosomes (for example, some viruses,endosomolytic amphipathic peptides), which enables said transport toproceed more effectively into one of the most PDD-sensitive cellcompartments—the nucleus. Lysosomes, for example, may serve as anothersensitive compartment—an object of targeted transport inside the targetcell. The PS carriers are used as various polymers, for exampleproteins, including recombinant polypeptides. Components which effecttarget cell recognition (binding with them) and direct transport insidethe cells by receptor-mediated endocytosis are intemalizable ligands, inparticular peptide hormones (insulin, somatotropin, prolactin, etc.),lectins (concanavalin A, ricin, wheat germ agglutinin, etc.), otherprotein (transferrin, ferritin) and protein-free (sugars, low-molecularhormones) components. Various molecules capable, when exposed to light,of generating active oxygen species (porphyrins, phthalocyanines, etc.)may be used as the PS.

For a targeted delivery of the PS into the eukaryotic cell nuclei, weuse a composition consisting of the following components: PS chlorin e₆,a carrier component—bovine serum albumin (BSA), a component for targetcell recognition and PS transport by receptor-mediatedendocytosis—intemalizable ligand, insulin, and a component capable ofeffective targeted transport of the PS within target cells—a karyophilicsequence of the SV40 large T-antigen ensuring targeted intracellulartransport of the composition into the nucleus. The present compositionhas the following structure: chlorin e₆, a karyophilic sequence andinsulin are covalently bound to a BSA carrier protein.

The composition comprises a chemically synthesized karyophilic sequenceof the SV40 large T-antigen (peptide PI 101). Peptide PI 101 comprises akaryophilic sequence.

Another composition has a similar structure but, in this case,βgalactosidase from E. coli serves as a carrier protein. A chimericprotein P10 is prepared in which a carrier protein (βgalactosidase) isfused with said component capable of effective targeted transport of thePS within target cells, namely with the karyophilic sequence of the SV40large T-antigen; a galactosidase part of protein P10 acts as a carrierand ensures an intracellular transport of the composition into thetarget cell nuclei; to this recombinant protein, the PS (chlorin e₆) andinsulin are covalently bound. It is shown that the presence of saidkaryophilic sequence leads to a significant enhancement of the targetcell PDD. However, even such composition is not able to be fullytransported into the cell nuclei since it comprises insulin and,therefore, such a composition, after being internalised into the targetcell by receptor-mediated endocytosis, appears to be enclosed withinendosomes. To leave endosomal vesicles, the composition should compriseone more component for a targeted transport of the PS inside the targetcells, namely a component enabling exit of the composition fromendosomes.

There has been also known a possibility for the disintegration ofendocytotic vesicles (for example, endosomes) by means of adenoviruses(Fitz-Gerald, J. D. P., Padmanabhan, R., Pastan, L, and Willingham, M.C. Adenovirus-induced release of epidermal growth factor- andPseudomonas toxin into the cytosol of KB cells during receptor-mediatedendocytosis. Cell, 32, 607-617, 1983).

A further composition is provided with an endosomolytic component—anonreplicating mutant virus type which enables exit of the compositionsfrom endosomes (human adenovirus, serotype 5, strain d1-312) (Jones, N.,Shenk, T. An adenovirus type 5 early gene function regulates expressionof other early viral genes. Proc. Natl. Acad. Sci. USA, 76, 3665-3669,1979).

A subsequent intracellular PS transport into the nuclei is carried outowing to the fact that the composition comprises a karyophilic sequence(as part of the protein P10 composition).

The present object is also solved owing to the enhancement of PDD byintroducing the PS to the cells in the composition having the ability ofa targeted transport of the PS within target cells; keeping them at atemperature of normal vital activity of the cells; and subsequentexposure of the cells to light.

As a result of such application of the composition, the PS is subjectedto targeted delivery into target cells, bound to specific receptors onthe target cells, transferred toward the interior of the target cells byreceptor-mediated endocytosis and transported into the mostPDD-sensitive cell compartments, and the PS photo-activation may be donemost efficiently. In so doing, the nuclei are employed as sensitivecompartments.

In accordance with the present invention, a method for causingphotodynamic damage to target cells includes the use of thosecompositions which comprise a photosensitiser, a photosensitiser carriercomponent, a component which enables target cell recognition andtransport of the photosensitiser toward the interior of the target cellby specific receptor-mediated endocytosis, and a component capable ofeffective targeted transport of the photosensitiser within target cells.The method for causing photodynamic damage to target cells also includesthe use of those compositions which have the ability of effectivetargeted transport into the cell nucleus and compositions having theability of releasing the photosensitiser from endocytotic compartments.

When solving the object in hand, it was taken into account that thephotodynamic effect of an overwhelming majority of PS (porphyrins,phthalocyanines) was due to the generation of the so-called “activeoxygen species” (singlet oxygen, some oxygen free radicals, etc.) knownto be properly toxic agents. It was also known that an average range ofsaid active oxygen species in the cell did not exceed a one hundredth ofa micron (Bekker, G. O., et al. Introduction into photochemistry oforganic compounds. 1976, p. 326). Because of this, cellular membranes inwhich the PS localise were the main target for the PS. The enhancementof PDD while decreasing an acting PS concentration was achieved by usingthe PS as part of the composition comprising components enabling atargeted transport of the PS into the most PDD-sensitive compartments oftarget cells, damage to which is critical for cell survival. Delivery ofthe PS to PDD-sensitive target cell compartments is carried out afterthe PS as part of the composition has been bound to the target cell andthen absorbed by the latter. To prepare compositions which could bebound to specific receptors of the target cells and then transportedinto said target cells by receptormediated endocytosis, conjugation ofthe PS with a ligand having such receptors on the target cell surface isperformed. Ligands such as, for example insulin, somatotropin and manyothers, after binding to their respective receptors on the cell surface,penetrate into the interior of the cells by receptor-mediatedendocytosis; and it is possible to accomplish a targeted intracellulartransport of the composition—receptor complex into the most sensitivecell compartments (Backer, J. M., Kahn, O. R., and White, M. F. Tyrosinephosphorylation of the insulin receptor during insulin-stimulatedinternalisation in rat hepatoma cells. J. Biol. Chem., 264, 1694-1701,1989).

If the component for a targeted transport is bound to a ligandpenetrating into the cell by receptor-mediated endocytosis, it ispossible, through a targeted delivery, that an intracellular PSlocalisation be replaced and said PS be delivered to more sensitive cellcompartments, thereby enabling decrease in an acting PS concentrationnecessary to be administered to achieve the effect.

A method for causing photodynamic damage to target cells usingcompositions for enhancing said photodynamic damage to target cells iscarried out as follows.

Components in the composition are synthesized; the composition isprepared to be suitable for a selected type of target cells; applied toan object containing target cells for PDD; keeping at a temperature ofnormal cellular vital activity, and a photosensitiser is subjected tophotoactivation.

The possibility to carry out the method for causing photodynamnic damageto target cells using compositions for causing photodynamic damage totarget cells of the photosensitiser is confirmed by the followingExamples.

EXAMPLE 1 Synthesis of (BSA)-(chlorin e₆)-(PI 101)-insulin CompositionStep 1. Preparation of chlorin e₆

Chlorin e₆ was prepared from nettle leaves (Urtica dioica L.) inaccordance with a standard method of Fischer (Fischer, H., Stern, A. DieChemie des Pyrrols. Leipzig: Akad. Verl., 2, 478S, 1940) withmodifications (Hynninen, P. H., Chlorophylls. IV. Preparation andpurification of some derivatives of chlorophylls a and b. Acta Chem.Scand., 27, 1771-1780, 1973).

Step 2. Synthesis of (BSA)-(chlorin e₆) Conjugate

BSA-chlorin e₆, conjugation was performed in Na-phosphate buffer (10 mM,pH 7.5) by means of cyclohexyl-3-(2-morpholinoethyl) carbodiimidemeto-4-toluenesulphonate (CDI) (Serva). The (BSA):(chlorin e₆):(CDI)ratio was 1:30:300. The reaction was carried out for 18 hours at 4° C.Thereafter, dialysis against the same buffer was performed.

Step 3. Activation of (BSA)-(chlorin e₆) Conjugate with3-maleimidebenzoyl Hydroxysuccinimide Ester (MBS)

To activate NH₂-groups of BSA for a further conjugation, the reactionwith MBS (Sigma) was carried out in the same Na-phosphate buffer at the(BSA):(MBS) ratio of 1:100. MBS was diluted in dimethyl formamide andadded to the (BSA)-(chlorin e₆) conjugate. A mixture was left for 1 hourat room temperature followed by removal of MBS residue with dialysisagainst Na-phosphate buffer (10 mM, pH 7.5; 0.5 mM EDTA).

Step 4. Synthesis of (BSA)-(chlorin e₆)-(PI 101)-insulin Composition

Peptide PI 101 at its C-end contains cysteine, via which SH-groupsconjugation of peptide with MBS-activated BSA amino groups may beperformed. When mixing the peptide with MBS-activated (BSA)-(chlorin e₆)conjugate under anaerobic conditions ((BSA)-(chlorin e₆) and peptideratio is 1:30), a covalent binding of the peptide to BSA occurs. Thereaction was carried out for 3 hours at room temperature followed byremoval of the peptide residue with dialysis against Na-phosphate buffer(10 mM, pH 7.5; 0.5 mM EDTA).

Step 5. Activation of Insulin by Sulfosuccinimide 4-(maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC)

Insulin and sulfo-SMCC (Pierce) were diluted in Na-phosphate buffer (10mM, pH 7.5) and mixed at a 1:1.5 ratio. The reaction was carried out for30 minutes at 37° C. Thereafter, dialysis against Na-phosphate buffer(10 mM, pH 7.5; 0.5 mM EDTA) was performed.

Step 6. Reducing SH-groups of BSA by Means of Dithiothreitol (DTT)

To (BSA)-(chlorin e₆)-(PI 101), DTT (Sigma) was added to a finalconcentration of 50 mM and incubated for 30 minutes at 37° C. followedby dialysis against Na-phosphate buffer (10 mM, pH 7.5; 0.5 mM EDTA) wasperformed.

Step 7. Preparation of (BSA)-(chlorin e₆)-(PI 101)-insulin Composition

Insulin and (BSA)-(chlorin e₆)-(PI 101) obtained as described in Steps 4and 5, respectively, were mixed under anaerobic conditions at the 15:1ratio. Incubation was carried out for 18 hours at 4° C. Thereafter, thecomposition was purified by column chromatography on Sephacryl S-300eluting with 25 mM HEPES, pH 7.5; 50 mM NaCl.

After the completion of all the conjugation steps, polyacrylamide gelelectrophoresis according to a method of Laemmli was performed todetermine molecular weight of the composition so obtained.

EXAMPLE 2 Determining Localisation of (BSA)-(chlorin e₆)-(PI 101)-insulin Composition in Human Hepatoma PLC/PRF/5 Cells

In order to reveal localisation of the (BSA)-(chlorin e₆)-(PI101)-insulin composition in cells, 2′,7′-dichlorofluorescein diacetatewas used. When incubating with cells at 37° C., this substancepenetrates into the cells and there it is deacetylated by intracellularesterases. If the production of active oxygen species occurs in theplace of 2′,7′-dichlorofluorescein localisation, then2′,7′-dichlorofluorescein, while interacting with these species,converts into fluorescent 2′,7′-dichlorofluorescein (Patel, A. K.,Hallet, M. B., and Campbell, A. K. Threshold responses in production ofreactive oxygen metabolites in individual neutrophils detected by flowcytometry and microfluorimetry. Biochem. J, 248, 173-180, 1987). Becauseit is known that the range of active oxygen species is very small, thecomposition localisation coincides with that of active oxygen speciesgenerated by this composition after irradiation of the cells and,therefore, in the presence of 2′,7′-dichlorofluorescein diacetate it ispossible to reveal both the place of the composition location in thecell and the place of generation of those toxic products which give riseto a PS-caused PDD.

Human hepatoma PLC/PRF/5 cells were incubated with 100 nM (BSA)-(chlorine₆)-(PI 101)-insulin composition for 18 hours at 37° C. in RPMI-1640medium containing 2 mg/ml BSA, 25 mM HEPES, pH 7.5. Upon the completionof incubation, cells were washed with the same medium, incubated for 5minutes with 2.5 μM 2′,7′-dichlorofluorescein diacetate, washed out fromthe latter and exposed to light of a slide projector followed by therecording of a fluorescence distribution in cells by means of cooled CDDcamera AT-200 (Photometrics). As can be seen in FIG. 1, the(BSA)-(chlorin e₆-(PI 101T)-insulin composition penetrates into the celland the nuclei much more effectively than the (BSA)-(chlorin e₆)-( PI101T)-insulin composition (peptide PI 101 T differs from peptide PI 101in that Thr⁷ was substituted for Lys⁷), wherein the transport of thecompositions into the cell is carried out via insulin receptors, whichis proved by the absence of fluorescence in cells during theirco-incubation with the composition and a 100-fold excess of freeinsulin.

EXAMPLE 3 Synthesis of (P10)-insulin-(chlorin e₆) Composition

For the preparation of the titled composition, a recombinant protein P10was used as the carrier protein (Ribs, H. -P., Jans, D. A., Fan, H., andPeters, R. The rate of nuclear cytoplasmic protein transport isdetermined by the casein kinase II site flanking the nuclearlocalisation sequence of SV40 large T-antigen. EMBO J., 10, 633-639,1991). Protein P10 is a product of chimeric gene in which a karyophilicsequence of the SV40 large T-antigen was attached to a sequence encodinga bacterial protein β-galactosidase from E. coli. A bacterialβ-galactosidase was used as control.

Step 1. Isolation of Proteins P10 and β-galactosidase

To prepare a chimeric gene P10 and β-galactosidase, E. colicell-expressed plasmids pDJ87 and pDJ148 were used, respectively.

For each protein, a corresponding E. coli culture was grown in LB-broth(Sigma) with Na-ampicillin and isopropyl-β-thiogalactoside (SibEnzyme)as a protein expression inducer. After centrifugation of bacterial massand ultrasound lysis, the suspension was re-centrifuged and supernatantapplied to affinity column using p-aminobenzyl1-thio-β-D-galactopyranoside (Sigma) as sorbent. After washing thecolumn with 20 mM Tris-HCl, pH 7.4; 10 mM MgCl₂; 1.6 M NaCl (Sigma); 10mM 2-mercaptoethanol (to remove other protein impurities), the proteinto be isolated was eluted with borate buffer (0.1 M, pH 10.05; 10 mM2-mercaptoethanol). The presence of protein in fractions was determinedby the colour reaction with o-nitrophenyl-β-D-galactopyranoside (Sigma)(Pardee, A. B., Jacob, F., and Monod, J. The genetic control andcytoplasmic expression of “inducibility” in the synthesis ofβ-galactosidase by E. coli. .l. Mol. Biol., 1, 165, 1959). Afterdialysis in phosphate buffer (10 mM, pH 7.0; 10 mM 2-mercaptoethanol),protein was concentrated by ultrafiltration. A protein concentration wasdetermined by absorption at 280 nm.

Step 2. Preparation of Chlorin e₆

Chlorin e₆ was prepared as described in Example 1.

Stems 3. Preparation of Aminochlorin

CDI and diaminohexane (Sigma) was added to chlorin e₆ in 5 mMNa-phosphate buffer, pH 7.5. The (chlorin e₆):(CDI):diaminohexane ratiowas 1:100:100. The reaction was carried out overnight at 4° C.Thereafter, excessive CDI and diaminohexane were removed by dialysis inNa-phosphate buffer (10 mM, pH 7.0).

Step 4. Preparation of Citraconated Insulin

To prepare conjugates with ligand having specific internalizedreceptors, insulin modified with citraconic anhydride was used.Insulin—citraconic anhydride interaction results in the protection ofthe terminal amino group of insulin which participate in the interactionwith receptors; therefore, the amino groups of lysine located in themiddle of polypeptide chain are able to be modified after insulincitraconation. Modification of insulin with citraconic anhydride wascarried out according to a method of Shechter (Shechter, Y.,Schlessinger, J., Jacobs, S., Chang, K. J., and Cuatrecasas, P.Fluorescent labeling of hormone receptors in viable cells: Preparationand properties of highly fluorescent derivatives of epidermal growthfactor and insulin. Proc. Natl. Acad. Sci. USA, 75, 2135-2139, 1978).

Step 5. Synthesis of (P 10)-(chlorin e₆) and (β-galactosidase)-(chlorine₆) Conjugates

CDI was also used for the conjugation of proteins with aminochlorin e₆.The reaction was carried out in Na-phosphate buffer (10 mM, pH 7.0) atthe protein: (aminochlorin e₆):(CDI) ratio of 1:30 300. The conjugateswere subjected to dialysis in order to be purified from unboundaminochlorin e₆ and excessive CDI.

Step 6. Synthesis of (P 10)-(chlorin e₆) and(β-galactosidase)-insulin-(chlorin e₆) Compositions

Insulin modified with citraconyl groups was covalently bound to(P10)-(chlorin e₆) and (β-galactosidase)-(chlorin e₆) conjugates bymeans of a bifunctional cross-linking agent,N-succinimidyl-3-(2-pyridildithio)-propionate (SPDP) (Sigma), accordingto the procedure disclosed by Jung et al. (Jung, G., Kohnlein, W., andLuders, G. Biological activity of antitumor protein neocarcinostatincoupled to a monoclonal antibody byN-succinimidyl-3-(2-pyridilthio)-propionate. Biochem. Biophys. Res.Commun., 101, 599-606, 1981). Insulin was taken at a concentrationexceeding 15-fold that of the conjugate, based on protein. Followingincubation and dialysis in Na-phosphate buffer (10 mM, pH 7.0),citraconyl groups were removed by acidification followed by extractionof compositions, as disclosed by Shechter et al. Thereafter, thecompositions were subjected to dialysis in Na-phosphate buffer (10 mM,pH 7.0).

To determine the number of bound insulin molecules per the compositionmolecule, electrophoresis was performed in a 5% polyacrylamide gelaccording to a method of Laemmli (Laemmli, U. K. Cleavage of structuralproteins during the assembly of the head of bacteriophage T4. Nature,227, 680-685, 1870). The protein:(chlorin e₆):insulin ratio in thecompositions was found to be 1:5:8.

EXAMPLE 4 PDD of Human Hepatoma PLC/PRF/5 cells Using(P10)-insulin-(chlorin e₆) Composition

Use of the composition in human hepatoma cells showed that the presencein the composition of a sequence permitting transport of the compositioninto the nucleus, enhanced to a greater extent the photodynamic actionof chlorin e₆ present in the composition. At the irradiation dose of12.3 kJ/m², the EC₅₀ value (concentration of a semi-maximal effect) wasfound to be 17 nM for the (P10)-insulin-(chlorin e₆) composition, 319 nMfor the control (β-galactosidase)-insulin-(chlorin e₆) composition and11,500 nM for free chlorin e₆. At the irradiation dose of 96 kJ/m², theEC₅₀ value was found to be 7.2. nM for the (P10)-insulin-(chlorin e₆)composition, 38 nM for the (β-galactosidase)-insulin-(chlorin e₆)composition and 350 nM for chlorin e₆. Survival rate of human hepatomaPLC/PRF/5 cells plotted against concentration of compositions andchlorin e₆ is depicted in FIGS. 2 and 3.

EXAMPLE 5 Method for Producing (P10)-insulin-(chlorin e₆)Composition+Endosomolytic Component (adenovirus) and(β-galactosidase)-insulin-(chlorin e₆) Composition+EndosomolyticComponent (adenovirus)

To escape compositions from intracellular endosomes into the cytosol, weused human adenovirus Ads d1-312. To this end, cells were incubated witha (P10)-insulin-(chlorin e₆) or (β-galactosidase)-insulin-(chlorin e₆)composition together with adenovirus. On the second day afterincubation, cells were washed out with RPMI-1640 medium supplementedwith 2 mg/ml BSA, 25 mM HEPES, pH 7.5 and incubated with saidcompositions or with said compositions in the presence of virus.Concentration of the compositions in the incubating medium was 20 nM,that of adenovirus—3.66×10¹⁰ virions/ml. In the experiments withadenovirus, a preliminary incubation for 1 hour at 4° C. was carried outto enable an efficient binding of virus and compositions to theirreceptors and subsequent simultaneous beginning of the intemalisationcycle of insulin and adenovirus receptors after the cell transfer to 37°C. Following 18 hour incubation at 37° C., the cells were washed usingthe same medium.

EXAMPLE 6 Localisation of Compositions (P10)-insulin-(chlorin e₆Composition+Endosomolytic Component (adenovirus) and(β-galactosidase)-insulin-(chlorin e₆)+Endosomolytic Component(adenovirus) in Human Hepatoma PLC/PRF/5 cells

During incubation of human hepatoma PLC/PRF/5 cells with the(P10)-insulin-(chlorin e₆) composition in the presence of adenovirus Ad5d1-312, the number of cells with a primary localisation of thecomposition in the nucleus increased by 148%, which was 3.5 times asmany as for the (β-galactosidase)-insulin-(chlorin e₆) composition (FIG.4).

INDUSTRIAL APPLICABILITY

The claimed composition for causing photodynamic damage to target cellsand method for causing photodynamic damage to target cells may beapplied in biotechnology, cellular and molecular biology when developingmethods in experimental medicine and pharmacology.

What is claimed is:
 1. A composition for causing photodynamic damage totarget cells, comprising: a photosensitizer; a carrier for thephotosensitizer; an endosomolytic component; a first component whichenables target cell recognition and transport of the photosensitizertoward the interior of the target cell by specific receptor-mediatedendocytosis; and a karyophilic moiety for targeted transport of thephotosensitizer to the nucleus of the target cells; wherein the carrieris conjugated to the karyophilic moiety, the photosensitizer and thefirst component.
 2. The composition according to claim 1, wherein saidendosomolytic component is adenovirus.
 3. The composition according toclaim 2, wherein said adenovirus is human adenovirus, type 5, straind1-312.
 4. The composition according to claim 1, wherein said carrierfor the photosensitizer is a polymer.
 5. The composition according toclaim 4, wherein said polymer is a protein.
 6. The composition accordingto claim 5, wherein said protein is bovine serum albumin.
 7. Thecomposition according to claim 5, wherein said protein is a recombinantpolypeptide.
 8. The composition according to claim 7, wherein saidrecombinant polypeptide is a bacterial β-galactosidase fused with saidkaryophilic moiety, said moiety being a karyophilic sequence of the SV40large T-antigen.
 9. The composition according to claim 1, wherein saidfirst component which enables target cell recognition and transport ofthe photosensitizer toward the interior of the target cell by specificreceptor-mediated endocytosis is an internalizable ligand.
 10. Thecomposition according to claim 9, wherein said internalizable ligand isa peptide hormone.
 11. The composition according to claim 10, whereinsaid peptide hormone is insulin.
 12. The composition according to claim1, wherein said photosensitizer is chlorin e6.
 13. A method for causingphotodynamic damage to target cells, comprising the steps of: contactingthe target cells with a composition of claim 1 so as to produce apopulation of treated cells by targeted transport of saidphotosensitizer into the target cells; keeping the population of treatedcells at a temperature of normal vital activity of cells for a period oftime; and exposing the population of treated cells to light, therebycausing photodynamic damage to the target cells.
 14. The methodaccording to claim 13, wherein said composition for causing photodynamicdamage is directed to the nucleus of the target cells by targetedtransport.
 15. The method according to claim 13, wherein saidcomposition for causing photodynamic damage is released from endocytoticcompartments.
 16. The composition of claim 1, wherein said karyophiliccomponent is covalently bound to the carrier.
 17. The composition ofclaim 1, wherein the first component is bound to the photosensitizer.